Raman analysis apparatus

ABSTRACT

A sample (14) is illuminated by light from a laser source (16), which is reflected to it by a dichroic filter (18) and passed through a microscope objective (20). The microscope objective (20) focusses a two dimensional image of the illuminated area onto a detector (22). On the way to the detector (22), the light passes through an interference filter (26), which selects a desired line from the Raman spectrum scattered by the sample (14). The filter (26) can be tuned to any desired Raman line by rotating it through various angles of incidence (Θ), about an axis (28) perpendicular to the optical axis.

BACKGROUND OF THE INVENTION

This invention relates to apparatus and methods in which the Ramaneffect is used to analyse a sample.

The Raman effect is a phenomenon in which a sample scatters incidentlight of a given frequency, into a frequency spectrum which has linescaused by interaction of the incident light with the molecules making upthe sample. Different molecular species have different characteristicRaman spectra, and so the effect can be used to analyse the molecularspecies present.

Analysis methods and apparatus using the Raman effect are described in apaper `Raman Microprobe and Microscope with Laser Excitation`, M Delhayeand P Dhamelincourt Journal of Raman Spectroscopy, 3 (1975) 33-43. Asample is irradiated with monochromatic light from a laser, and thescattered light is passed through a monochromator in order to select aparticular line of the resulting Raman spectrum. The paper describesboth a microprobe, in which light from a single illuminated point or aline on the sample is passed through the monochromator, and a microscopein which an area is illuminated and an integral, two dimensional imageof that area is passed through the monochromator. The microprobe has thedisadvantage that in order to obtain a two dimensional image, it isnecessary to scan a series of points or lines over the area of thesample, so that building up the required image is complex and may take arelatively long time. The microscope obviously does not suffer from thisdisadvantage,.but the optics of the monochromator require substantialmodification in order to pass a two dimensional image.

Specifically, a conventional monochromator has an optical system whichfocuses an image of the illuminated point or line of the sample onto anentrance slit; and a further optical system which focuses an image ofthe entrance slit onto an exit slit. Between the entrance slit and theexit slit there is a dispersive device such as a diffraction grating (orcommonly two or three such gratings in series). The dispersive devicehas the effect of splitting an incoming polychromatic light beam into arange of angles, depending on frequency. Because of the dispersion, theposition of the exit slit relative to the diffraction grating selectsthe desired spectral line to be investigated. The monochromator can betuned to different spectral lines by moving the exit slit, or moreconveniently by an arrangement in which the diffraction grating isrotated relative to the exit slit. Because the frequencies of thespectrum are separated by a dispersive process, it is obvious that goodfrequency resolution requires narrow slits. Since the image of thesample is focused on the slits, this is the reason why this conventionalmonochromator arrangement cannot observe a two dimensional area of thesample. If a wider slit were used in order to pass a two dimensionalimage, it would pass a range of frequencies. Because any given spectralline has a finite width, the result is a blurred image of any givenpoint on the sample, and if one attempts to form an image in twodimensions, the blurred image of one point in the sample will overlapwith the blurred image of an adjacent point, resulting in a veryconfused image (poor spatial resolution in addition to degradedfrequency resolution).

The modified optical system used in the above paper in order to providean integral two dimensional image, forms an image of the sample on thediffraction grating of the monochromator, instead of on the entrance andexit slits. At the entrance and exit slits, there are formed images ofthe exit aperture of an optical microscope which views the area of thesample which is to be imaged. By these means (in theory) one can pass anintegral two dimensional image of the area of the sample through amonochromator with arbitrarily narrow entrance and exit slits. However,the aperture size of the entrance slit governs the amount of light whichis collected and focused onto the grating; while the aperture size ofthe exit slit governs the amount of light which is collected from thegrating and focused to produce the resulting two dimensional image whichis detected. In the result, therefore, if one makes the entrance andexit slits narrow, to improve the frequency resolution, then theintensity of the resulting image is extremely low and difficult todetect. This is exacerbated by the fact that the desired Raman spectraare already of very low intensities, and cannot be increased merely byincreasing the incident illumination of the sample by the laser, sinceincreased laser power is likely to destroy the sample. Accordingly,commercial Raman analysis devices have tended to be of the scanningmicroprobe type, rather than a Raman microscope in which an integral twodimensional image is formed.

SUMMARY OF THE INVENTION

The present invention is based upon the realisation by the inventor thatif one uses a non-dispersive filter rather than a dispersivemonochromator, there is no need for narrow entrance and exit slits toprovide adequate frequency resolution. The above-noted paper does indeedbriefly suggest that the optical system could be simplified by using afixed wavelength interference filter, with the use of a tunable dyelaser in order to tune the system to different Raman lines, but theauthors state that this experiment did not succeed.

One aspect of the present invention provides Raman analysis apparatuscomprising:

means for illuminating a sample so as to produce therefrom a Ramanspectrum,

tunable means for selecting a desired frequency of said Raman spectrumreceived from the sample, and

a detector for detecting the light selected by the tunable means,

characterised in that said tunable means comprises non-dispersive filtermeans for selecting the desired frequency without splitting differingfrequencies into a range of angles.

Such apparatus may form either a Raman microscope or a Raman microprobe.

Preferably the filter means comprises an interference filter, such as adielectric filter, and preferably it is tuned by altering the angle ofincidence of the light scattered by the sample onto the filter. This maybe done by making the filter rotatable about an axis perpendicular tothe optical axis, to the required angles of incidence.

In another aspect, the invention provides a method of analysing a sampleusing such apparatus.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention will now be described by way ofexample, with reference to the accompanying drawings, wherein:

FIG. 1 is a graph showing a Raman spectrum,

FIG. 2 is a schematic diagram of a simple embodiment of the invention,and

FIGS. 3 and 4 are schematic diagrams illustrating further embodiments.

DESCRIPTION OF PREFERRED EMBODIMENTS

The Raman effect will first be briefly explained. If a sample isilluminated by monochromatic light of frequency ω_(L), eg. from a laser,light will be scattered. The graph of FIG. 1 shows the frequency ω ofthe scattered light, against intensity I. Most of the light is scatteredby Rayleigh scattering, giving a peak 12 at frequency ω_(L), but smallquantities of light are scattered at other frequencies because ofvarious effects, including fluorescence and the Raman effect. The Ramaneffect is caused by the various natural frequencies of interatomicvibration within the molecules which make up the sample. FIG. 1 shows anumber of Raman lines 10, at frequencies (ω_(L) -ω_(o)), where ω_(o) isthe natural frequency of a molecular vibration. Any given species ofmolecule will have a characteristic set of Raman lines, which can beused to identify the composition of the illuminated surface of thesample (or of the interior of a transparent sample). Such use of theRaman effect for analysis of a sample is already well understood.Performing an analysis involves measuring the frequencies and relativeheights h of the peaks 10, in order to be able to match the observedspectrum with the known spectra of various molecules.

FIG. 2 shows schematically an apparatus according to the invention forperforming such analysis. A sample 14 is illuminated over a twodimensional area of its surface by a monochromatic laser source 16. Thelaser beam is reflected from the source to the sample by a dichroicinterference filter (in this case a multilayer dielectric filter) 18,which is designed to transmit all frequencies except the very narrowline of the monochromatic laser source 16. A half-silvered mirror couldbe used in place of the filter 18, though this entails a loss of lightlevel. The laser beam also passes through a microscope objective 20,which is represented in the drawing as a single lens, but which may wellcomprise a system of several lenses in well known manner. The lasersource 16 may use a laser of any suitable type, e.g. a He-Ne laser, butif it is an ion laser it should include an appropriate filter to removeplasma lines.

The microscope objective 20 focuses a two dimensional image of theilluminated area 14 of the sample onto a camera 22. The camera 22 ispreferably a charge coupled device (CCD), cooled to cryogenictemperatures (eg. by liquid nitrogen or by Peltier cooling) in order toeliminate dark current and thereby improve the signal to noise ratio ofthe apparatus. The use of CCD's in conventional Raman spectroscopy isdescribed in a paper `Multichannel Raman Spectroscopy with a Cooled CCDImaging Detector`, D N Batchelder, ESN--European Spectroscopy News, 80(1988), pages 28,32,33. The output of the CCD 22 is taken to a computer24, which is used both for data collection from the CCD and for analysisof the resulting images. Other cameras could of course be used in placeof the CCD 22, such as an intensified video camera.

Between the filter 18 and the CCD 22, there is placed a second linefilter 26. This is again a dielectric interference filter. Unlike thefilter 18, however, the filter 26 is arranged to transmit only thefrequency (ω_(L) -ω_(o)) of a Raman line of interest, and to reject allother frequencies. In the rejection of other frequencies, it is ofcourse assisted by the filter 18 which has already rejected much of theexciting frequency ω_(L).

Thus, the image which is focused on the CCD 22 is a two dimensionalimage of the scattering at the selected Raman frequency ω_(L) -ω_(o),over the illuminated area of the sample 14. This image has excellentspatial resolution, limited only by the quality of the microscopeobjective 20 and the resolution of the CCD 22. The computer 24 isprogrammed to record the intensity of the selected Raman peak 10 foreach pixel of the two dimensional image received by the CCD.

The filter 26 is arranged for pivotal movement about an axis 28,perpendicular to the optical axis, through an angle Θ. This allows us tomake use of a property of such interference filters, namely that thefrequency of the line transmitted by the filter varies with the angle ofincidence Θ. Thus, by adjusting the angle 8, the apparatus can be tunedto select different peaks 10 within the spectrum of FIG. 1. Also, bytuning first to a frequency A on one of the peaks 10, and taking anintensity reading for each pixel of the image, then tuning to anadjacent frequency B and repeating the intensity measurements, thecomputer 24 can calculate the height h of the peak, simply bysubtracting one reading from the other, for each pixel of the image.

The filter 26 is tuned by turning it to the required angle Θ by means ofa rotary drive 30. At its simplest, this may simply comprise a manualadjustment device, such as a knob, optionally with a pointer to a scaleindicating the angle Θ (which may be calibrated in terms of the wavenumber in cm⁻¹). Preferably, however, the drive means 30 is a motorisedrotary drive, eg. a stepping motor. Optionally, this may be controlledby the computer 24, as indicated by a broken line 32. This enables thecomputer 24 to be programmed with a pre-determined analysis procedure,so that the analysis can proceed entirely automatically. In such a mode,the computer 24 adjusts the angle of incidence 8 to a firstpre-determined Raman frequency, gathers and stores data from the CCD 22relating to the two dimensional image at that Raman frequency, and thenrepeats this procedure for each of a number of different pre-determinedRaman frequencies. The computer programme can then automatically performany desired analysis, such as the simple analysis described above ofsubtracting the readings at a frequency A from those at a frequency B.It can also compare the Raman spectrum it has detected for each pixel inthe image with data pre-stored in a data bank relating to the knownRaman spectra of different molecular species, so as to make adetermination as to which species are present.

The rotary drive 30 may include not only a motor controlled by thecomputer 24, but also an angular position encoder which providesfeed-back of the current angular position Θ to the computer, in order toprovide servo control of the angle Θ.

Alternatively, if desired, the rotary drive for the filter 26 maycomprise an oscillator, which oscillates the filter through a range ofangles 0. This range of angles may be chosen so as to include the twopoints A and B in FIG. 1. The computer 24 is then programmed to samplethe output of each pixel of the CCD 22 over the range of oscillation,and to calculate the peak to peak value of the resulting signal for eachpixel. This gives an easy method of determining the height h of the peak10.

The interference filters 18, 26 can be obtained commercially, forexample from Omega Optical Inc., Brattleboro, Vt., USA. The filter 18may be obtained off the shelf for any of a variety of frequencies ω_(L)corresponding to the commonly used laser excitation frequencies. Thefilter 26, however, has to be specifically designed to transmit thedesired Raman frequency (or rather, range of Raman frequencies varyingwith angle Θ). However, the design and supply of such filters to anydesired frequency is a commercially available service from companiessuch as Omega Optical Inc.

It will be noted that the filters 18, 26 being interference filters, arenot dispersive (that is, they do not disperse light of varyingfrequencies into a corresponding range of directions). It is thisfeature which gives the present apparatus its good spatial resolution,albeit at the expense of some degree of frequency resolution whencompared with conventional dispersive monochromator apparatus. This isachieved without significant reduction of the intensity of the Ramanline being investigated. With currently available commercialinterference filters, it may be necessary to provide perhaps 4 or 5interchangeable filters 26, in order to cover the range of frequenciesof interest. This is because an individual such filter cannot be tunedover the entire likely range of frequencies by altering the angle Θ. Tothis end, the filter 26 is preferably arranged in an appropriate mountso as to facilitate its removal and replacement. Alternatively, suchremoval and replacement may be performed by an automatic changing deviceunder the control of the computer 24.

Other non-dispersive filters may be used in place of the dielectricfilter 26. For example, a Fabry-Perot interferometer may be used as sucha filter, and may be tuned over the desired range of frequencies byadjusting the spacing of its plates, or by altering the refractive indexof the medium between the plates (eg. by adjusting the pressure of agaseous medium). As the filter 18, one could use a crystalline colloidalBragg diffraction device as described by Asher et al., Spectroscopy,Vol. 1 No. 12, 1986, pp 26-31. Such devices are available from EG & GPrinceton Applied Research, Princeton, N.J., USA.

FIG. 3 shows an embodiment of the invention with an alternative opticalarrangement. Here, the sample 14 is placed in the focal plane of themicroscope objective 20. Thus, the microscope objective 20 does notdirectly focus an image of the sample onto the CCD 22, but insteadproduces a parallel beam of light in respect of any given point on thesample. This is focused by a further lens or lens system 34 to producethe required two dimensional image on the CCD. The additional lens 34 isplaced after the filter 26 which is tuned to the desired Raman frequencyas described above in respect of FIG. 2.

It will be appreciated that since the sample 14 is now in the focalplane of the microscope objective 20, the parallel beam of lightproduced by the laser source 16 would illuminate only a single point onthe sample, and not provide illumination over a two dimensional area asrequired. This is overcome by providing a convergent or divergentillumination beam, so that the laser source 16 is focused either behindor in front of the surface of the sample 14. The angle of divergence orconvergence of the incident beam is matched to the size of the area ofthe sample which is to be illuminated. As an example, FIG. 3 illustratesthat the parallel laser light is formed into a convergent beam by aconvex lens 38. Although not essential, the Figure illustrates that thisconvergent beam is focused on a central point of the filter 18. In thiscase, the filter 18 may if desired be provided with a small metallisedspot at its central point, to improve the reflectance of the incidentlaser beam and prevent damage to the filter. In this case, it isdesirable to include a filter in the laser source 16 to reject anyspurious radiation from the laser source.

FIG. 3 also shows an optional further filter 36 placed in the light pathafter the filter 18. Like the filter 18, the filter 36 is arranged toreject only the excitation frequency ω_(L). By providing betterrejection of the excitation frequency, the signal to noise ratio of theapparatus is improved.

The apparatus described above is arranged as a Raman microscope, passinga two dimensional image. However, it can easily be modified for use as aRaman microprobe. This is done by providing a parallel input laser beam,which is focussed to a point on the sample 14 by the microscopeobjective 20. The resulting image is focussed onto a small group ofpixels on the CCD 22. The computer 24 averages the outputs of thesepixels. This gives the intensity of the Raman line selected by thefilter 26. The apparatus can be tuned to the various Raman lines ofinterest by rotating the filter 26, as previously. The sample 14 may, ifdesired be mounted on a table which is slidable in two orthogonaldirections X and Y, so that the spot of this Raman microprobe can bescanned across the area of the sample, in a generally conventionalmanner.

The apparatus described can also easily be modified for analysing thefluorescence of the sample. This is achieved simply by replacing thetunable filter 26 with a more broad band filter appropriate forfluorescence work.

A microprobe as described above with an X-Y scanning arrangement is alsouseable for contour scanning work, to determine the shape and dimensionsof the object being studied. For such work, the microscope objective 20is arranged to produce a slightly de-focussed spot on the object 14. Thedistance of the illuminated spot on the object 14 from the microscopeobjective 20 then governs the size of the image produced on the CCD 22(i.e. the number of pixels illuminated by this image). The computer 24is programmed to determine the size of the image. As X-Y scanning takesplace, the size of the image on the CCD 22 will vary with the localheight of the illuminated portion of the object 14, and can bedetermined by the computer. This makes a powerful analysis tool whichcan determine both the shape and contour and also the local compositionof the object 14.

In either of the embodiments shown in FIGS. 2 and 3, when the filter 26is rotated the image is displaced slightly because of the refraction inthe glass substrate. This can be corrected mechanically by having acounter-rotating piece of glass of the same optical thickness in thebeam, as discussed below in relation to FIG. 4, or by shifting the imageappropriately by means of software in the computer.

If desired, the filter 26 can be removed so as to produce an ordinaryoptical image of the sample on the CCD. This enables features found inthe Raman images to be referenced to the position as seen in theordinary optical image. The sample can be illuminated with an additionalwhite light source for this purpose, if desired.

In another modification, the filter 18 may be arranged to transmit thelaser frequency ω_(L) and reflect all other frequencies, instead of theinverse arrangement shown in FIGS. 2 and 3. In this case, the light fromthe source passes through the filter 18 in a straight line to the sample14, and the filter 26, CCD, etc. are arranged at right angles to thisstraight line such that the optical path from the sample 14 to the CCD22 is reflected through an angle of 90° at the filter 18.

FIG. 4 illustrates a practical embodiment of the apparatus according tothe invention. The same reference numerals as in FIGS. 2 and 3 have beenused to denote similar features. The laser input passes through a lenssystem 40, which may include a spatial filter (e.g. a pinhole 41) toimprove beam quality. The beam is reflected by a mirror 42 to the filter18. The microscope objective 20 is provided as part of a conventionaloptical microscope 48, and a mirror 46 is provided to reflect light toand from the objective 20. The mirror 46 can be removed to permitordinary use of the optical microscope 48, e.g. to permit setting-up andordinary optical examination of the sample 14. For these purposes, themicroscope 48 has a source 50 of white light for illuminating thesample. The sample 14 is placed upon a movable table 52. As shown, thisis simply movable in the vertical direction for focussing purposes; butas discussed above a table can be provided which can also be scanned inhorizontal X and Y directions.

An optional polarising filter 44 is provided in the path of the lightfrom the objective 20 to the CCD 22. This can be inserted into orremoved from the optical path, and is rotatable about the optical axisto vary the direction of polarisation. This enables investigation of thepolarisation state (if any) of the Raman line under investigation, whichcan yield additional useful information when analysing some materials.The polarising filter 44 may be rotated under the control of thecomputer 24, if desired, for automatic analysis.

FIG. 4 shows the rotatable, tunable filter 26 mounted on a rotatablewheel 27 for control either manually or by the computer 24 via therotary drive 30. The figure also shows a second such rotatable wheel 53for mounting a second filter 54. The wheel 53 is linked to the wheel 27,e.g. by a wire 55, so that it rotates with the wheel 27 but in theopposite direction. In a simple arrangement, the filter 54 is a plainpiece of glass, which corrects for the slight displacement of the imageon the CCD 22 caused by refraction as the filter 26 rotates. Desirably,however, the filter 54 is another dielectric filter similar to thefilter 26, tuned to a slightly different centre frequency but having anoverlapping pass band. The result of passing the light through the twofilters 26,54 having an overlapping pass band is to enable even moreselective tuning to a particular Raman line of interest.

The two filters 26,54 may be so linked that they always have an equalbut opposite angle of incidence Θ to the optical axis. In this case, twodifferent filters having two slightly different centre frequencies willbe required. Alternatively, however, the two filters may be identical,but the mounting of one of them on the corresponding wheel 27,53 isadjusted so that the two filters have slightly different angles ofincidence. This achieves the same effect by reason of the tunable natureof the filters. A further possibility is illustrated in broken lines: inplace of the mechanical wire link 55, the wheel 53 has its own rotarydrive 56, similar to the drive 30, controlled by the computer 24 via aline 58. Appropriate programming of the computer 24 can therefore seteach wheel 27,53 independently to any desired angles of incidence.Producing the desired counter-rotation of one wheel relative to theother, and setting the desired degree of overlapping of the pass bandsof the filters 26,54 is a straightforward programming task.

We claim:
 1. Raman analysis apparatus comprising:means for illuminatinga sample so as to produce therefrom a Raman spectrum, non-dispersivetunable filter means for selecting a desired frequency of said Ramanspectrum received from the sample without splitting differingfrequencies into a range of angles, said tunable filter means beingcontinuously tunable over at least a band of said spectrum including aplurality of Raman lines, and a detector for detecting the lightselected by the tunable filter means.
 2. Apparatus according to claim 1,wherein the filter means comprises an interference filter.
 3. Apparatusaccording to claim 2 wherein the filter is a multilayer dielectricfilter.
 4. Apparatus according to claim 1, wherein the filter means istunable by altering the angle of incidence of the light scattered by thesample onto the filter means.
 5. Apparatus according to claim 4, whereinthe filter means is rotatable about an axis perpendicular to an opticalaxis of the apparatus.
 6. Apparatus according to claim 5, includingmeans for counteracting refraction by the filter means as the filtermeans is rotated.
 7. Apparatus according to claim 4, wherein thenon-dispersive filter means includes two rotatable filters havingdifferent centre frequencies but overlapping pass bands.
 8. Apparatusaccording to claim 1, including a further filter for removing light ofan illuminating frequency emitted by the illuminating means from thelight received from the sample.
 9. Apparatus according to claim 1,wherein the illuminating means illuminates an area of the sample, and acorresponding two dimensional image is produced on the detector. 10.Apparatus according to claim 1, wherein the detector is a charge coupleddevice.
 11. Apparatus according to claim 1, wherein the detector is acharge coupled device.
 12. Apparatus according to claim 1, including aspatial filter in the path of light between the illuminating means andthe sample.
 13. Apparatus according to claim 12, wherein the spatialfilter comprises a pinhole.
 14. A Raman analysis methodcomprising:illuminating a sample so as to produce therefrom a Ramanspectrum, using non-dispersive tunable filter means for selecting adesired frequency of said Raman spectrum received from the samplewithout splitting differing frequencies into a range of angles, saidtunable filter means being continuously tunable over at least a band ofsaid spectrum including a plurality of Raman lines, and detecting thelight selected by the tunable filter means.
 15. Raman analysis apparatuscomprising:means for illuminating an area of a sample so as to producetherefrom a Raman spectrum, non-dispersive tunable filter means forselecting a desired frequency of said Raman spectrum received from thesample without splitting differing frequencies into a range of angles,said tunable filter means being continuously tunable over at least aband of said spectrum, and imaging means for producing a two dimensionalimage of said area of said sample, said image being formed with lightselected by said tunable means, and detecting means for detecting saidtwo dimensional image.
 16. Apparatus according to claim 15, wherein thefilter means comprises an interference filter.
 17. Apparatus accordingto claim 16, wherein the filter means is a multilayer dielectric filter.18. Apparatus according to claim 15, wherein the filter means is tunableby altering the angle of incidence of the light scattered by the sampleonto the filter means.
 19. Apparatus according to claim 18, wherein thefilter means is rotatable about an axis perpendicular to an optical axisof the apparatus.
 20. Apparatus according to claim 19, including meansfor counteracting refraction by the filter means as the filter means isrotated.
 21. Apparatus according to claim 15, wherein the nondispersivefilter means includes two filters having different centre frequenciesbut overlapping pass bands.
 22. Apparatus according to claim 15,including a further filter for removing light of an illuminatingfrequency emitted by the illuminating means from the light received fromthe sample.
 23. Apparatus according to claim 15, including a spatialfilter in the path of light between the illuminating means and thesample.
 24. Apparatus according to claim 23, wherein the spatial filtercomprises a pinhole.
 25. A Raman analysis method comprising:illuminatingan area of a sample so as to produce therefrom a Raman spectrum, usingnon-dispersive tunable filter means for selecting a desired frequency ofsaid Raman spectrum received from the sample without splitting differingfrequencies into a range of angles, said tunable filter means beingcontinuously tunable over at least a band of said spectrum, andproducing a two dimensional image of said area of said sample, saidimage being formed with light selected by said tunable means.